WO2021062406A1 - Promédicaments à base de cytokine et promédicaments doubles - Google Patents

Promédicaments à base de cytokine et promédicaments doubles Download PDF

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WO2021062406A1
WO2021062406A1 PCT/US2020/053155 US2020053155W WO2021062406A1 WO 2021062406 A1 WO2021062406 A1 WO 2021062406A1 US 2020053155 W US2020053155 W US 2020053155W WO 2021062406 A1 WO2021062406 A1 WO 2021062406A1
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prodrug
moiety
subunit
antibody
cancer
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PCT/US2020/053155
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English (en)
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Yuefeng Lu
Chunxiao YU
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AskGene Pharma, Inc.
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Priority to CA3153785A priority Critical patent/CA3153785A1/fr
Priority to CN202080064459.1A priority patent/CN114401997A/zh
Priority to EP20801423.3A priority patent/EP4034551A1/fr
Priority to AU2020353235A priority patent/AU2020353235A1/en
Priority to JP2022519172A priority patent/JP2022549344A/ja
Priority to US17/762,864 priority patent/US20220356221A1/en
Publication of WO2021062406A1 publication Critical patent/WO2021062406A1/fr

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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Definitions

  • Interleukin 12 plays important roles in immunity (Watford et al., Cytokine Growth Factor Rev. (2003) 14(5):361-8). It stimulates interferon gamma release and activates T cells and natural killer (NK) cells (Del Vecchio, Clin Cancer Res. (2007) 13(16):4677-85).
  • IL- 12 is comprised of two subunits, p40 and p35.
  • IL-12 receptor consists of two subunits, IL-12 receptor beta 1 (IL-12R ⁇ 1) and beta 2 (IL-12R ⁇ 2).
  • IL-12R ⁇ 1 Deficiency in IL-12R ⁇ 1 impairs the IL-12 signal pathway and may lead to bacterial infection and even autoimmunity such as Sjögren’s Syndrome (Sogkas et al., Front Immunol. (2017) 8:885).
  • IL-12 has previously been explored as a potential therapy to treat cancer (Colombo and Trinchieri, Cytokine Growth Factor Rev. (2002) 13(2):155-68; Del Vecchio, supra; Lasek et al., Cancer Immunol Immunother. (2014) 63(5):419- 35).
  • Several clinical trials on IL-12 therapy showed limited efficacy, potentially due to its narrow therapeutic index (Lasek et al., supra).
  • IL-2 another important cytokine, plays a central role in lymphocyte generation, survival and homeostasis. It has 133 amino acids and consists of four antiparallel, amphiphatic alpha-helices that form a quaternary structure indispensable of its function (Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13). [0005] IL-2 acts by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits. Association of the ⁇ (CD25), ⁇ (CD122), and ⁇ (CD132) subunits results in a trimeric, high-affinity IL-2R.
  • IL-2R IL-2 receptors
  • Dimeric IL-2R consisting of the ⁇ and ⁇ subunits is termed intermediate- affinity IL-2R.
  • the ⁇ subunit forms the monomeric low affinity IL-2R.
  • the dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, both the dimeric and trimeric IL-2R variants are able to transmit signal upon IL-2 binding (Minami et al, Annu Rev Immunol. (1993) 11:245-68).
  • Trimeric IL-2R is expressed by CD4 + FoxP3 + regulatory T (Treg) cells. They are also transiently induced on conventional activated T cells, whereas in the resting state these cells express only dimeric IL-2R. Treg cells consistently express the highest level of CD25 in vivo (Fontenot et al., Nature Immunol. (2005) 6:1142-51).
  • IL-2 has been made to reduce the side effects of IL-2. See, e.g., U.S. Pats.6,955,807 and 9,428,567 and EP2639241B1. Pegylated IL-2 with reduced affinity to the high-affinity trimeric IL-2R has also been disclosed (US20140328791).
  • IL-2 muteins have been fused to antibodies, such as PD-L1 antibody (e.g., WO 2017/220989) and CEA antibody (e.g., Klein et al., Oncoimmunology (2017) 6(3):e1277306; U.S. Pat.9,206,260).
  • the CEA antibody was maturated from an original mouse CEA antibody PR1A3 (U.S. Pat. 8,642,742). While the antibodies may target the IL-2 muteins to the tumor site, the amount of antibody-IL-2 fusion at the tumor site is likely only a small portion of the administered dose, with a large part of the administered dose still in the circulation.
  • the circulating IL-2 would bind to the so-called “PK sinkers” such as IL-2Rs on immune cells localized in immune organs, e.g., lymph nodes and the spleen. Therefore, even with the tumor-targeting specificity of the antibody, the total amount of the IL-2 that can be dosed would still be limited.
  • IL-15 is a cytokine with structural similarities to IL-2.
  • IL-15 is secreted by mononuclear phagocytes and other immune cells following viral infection.
  • IL-15 induces proliferation of NK and other immune cells and is involved in the killing of virally infected cells and cancer cells.
  • IL-15 binds to the IL-2R ⁇ / ⁇ complex, the intermediate-affinity receptor, with a K D of about 1 nM (Giri et al., EMBO J. (1994) 13:2822-30).
  • IL-15 binds to IL- 15 receptor (IL-15R) ⁇ with a much higher affinity (KD ⁇ 0.05 nM).
  • IL-15R ⁇ can associate with the IL-2R ⁇ / ⁇ complex to form an IL-15-specific, functional high-affinity receptor ( ⁇ ) (Minami et al., Annu Rev Immunol. (1993) 11:245-67; Giri et al., J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., Eur Cytokine Netw. (2000) 11:207-15).
  • the extracellular region of IL-15R ⁇ contains a Sushi domain, which is a common motif in protein-protein interaction.
  • the present disclosure provides an IL-12 prodrug or (“prodrug”) comprising an IL-12 cytokine moiety, a masking moiety (M), a dimeric carrier moiety (C), and at least one peptide linker, wherein the IL-12 cytokine moiety comprises an IL-12 p40 subunit (p40) and an IL-12 p35 subunit (p35), the masking moiety binds to the IL-12 cytokine moiety and inhibits a biological activity of the IL-12 cytokine moiety, and the masking moiety is fused to one polypeptide chain of the dimeric carrier moiety, optionally via a peptide linker, and the IL-12 cytokine moiety is fused to the
  • the IL-12 subunit p40 has an amino acid sequence at least 95% identical to that of SEQ ID NO: 5.
  • the IL-12 subunit p35 has an amino acid sequence at least 95% identical to that of SEQ ID NO: 6.
  • at least one peptide linker is a cleavable peptide linker. In other embodiments, at least one peptide linker is a non-cleavable peptide linker.
  • the masking moiety is a first masking moiety (MM1) that is fused to the carrier moiety or to the IL-12 p40 or p35 subunit, optionally via a cleavable peptide linker.
  • the first masking moiety (MM1) binds to IL-12 p40 subunit and reduces or inhibits the binding of IL-12 to IL-12 receptor ⁇ 1 (IL-12R ⁇ 1).
  • the masking moiety is selected from the extracellular domain of IL-12 receptor ⁇ 1 (IL-12R ⁇ 1 ECD) or a fragment thereof, a p40-binding peptide discovered from screening of a peptide library, and a single chain Fv (scFv) or Fab domain of an antibody that binds to p40.
  • the masking moiety is a scFv or Fab domain of an antibody that binds to p40.
  • the masking moiety comprises a scFv that binds to p40, wherein the scFv comprises an amino acid sequence at least 99% identical to SEQ ID NOs: 7-11 and 12.
  • the prodrug further comprises a second masking moiety (MM2) that is fused to the carrier moiety or to the IL-12 p40 or p35 subunit, optionally via a cleavable peptide linker.
  • MM2 second masking moiety
  • IL-12R ⁇ 2 IL-12 receptor ⁇ 2
  • the second masking moiety is selected from the extracellular domain of IL-12 receptor ⁇ 2 (IL-12R ⁇ 2 ECD) or a fragment thereof, a p35 or p40-binding peptide discovered from screening of a peptide library, and a single chain Fv or Fab domain of an antibody that binds to p35 or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2.
  • the second masking moiety comprises the extracellular domain (ECD) of IL-12 receptor beta2 (IL-12R ⁇ 2) or a fragment thereof, wherein the IL-12R ⁇ 2 ECD or a fragment thereof comprises an amino acid sequence at least 95% identical to SEQ ID NO: 17, 18, or 19.
  • the second masking moiety comprises a single chain Fv (scFv) or Fab domain of an antibody that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; wherein said scFv of Fab comprises the same light chain CDRs and heavy chain CDRs as derived from IL-12 antibody PMA204, Antibody 1, Antibody 50, Antibody 68, Antibody 80, or Antibody 136.
  • the carrier is an antibody or an antigen-binding moiety that binds to a target expressed on the surface of a cancer cell.
  • the antibody or antigen-binding moiety binds to an antigen selected from 5T4, Claudin 18.2, EGFR, EGFR type III, GPC3, TROP-2, mesothelin, PSMA, CMET, DLL-3, and BCMA.
  • the carrier moiety is selected from an Fc domain and an antibody.
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, and wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a.
  • F1-PL1-A-PL2-B F2-CL1-MM2-CL2-MM1; and b.
  • PL1 and PL2 are peptide linkers;
  • CL1 and CL2 are cleavable peptide linkers;
  • MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1;
  • MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2;
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-B, F2-PL3-MM2-CL-MM1; and b.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1, PL2, and PL3 are peptide linkers
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-CL1-MM2, F2-PL2-B-CL2-MM1; and b.
  • Fc domain carrier moiety
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-CL-MM2, F2-PL2-B-PL3-MM1; and b.
  • F1-PL1-A-PL2-MM1, F2-PL3-B-CL-MM2 wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1, PL2, and PL3 are peptide linkers; CL is a cleavable peptide linker; MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL- 12R ⁇ 1; MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • Fc domain carrier moiety
  • PL1, PL2, and PL3 are peptide linkers
  • CL is a cleavable peptide linker
  • MM1 is the first masking moiety
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-B, F2-CL-M; and b.
  • F1-PL1-B-PL2-A, F2-CL-M wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1 and PL2 are peptide linkers
  • CL is a cleavable peptide linker
  • M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12
  • A is an IL-12 p35 subunit
  • B is an IL-12
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-M, F2-PL3-B; and b.
  • F1-PL1-B-PL2-A, F2-PL3-M wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1, PL2, and PL3 are peptide linkers; M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1, PL2, and PL3 are peptide linkers
  • M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12
  • A is an IL-12 p35 subunit
  • B is an IL-12 p40 subunit.
  • the carrier moiety comprises an Fc domain, which comprises a first and a second polypeptide chain, wherein the polypeptide chains comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-M, F2-PL2-A-SS-B; and b.
  • F1-CL, F2-PL1-A-SS-B wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit; and SS is a disulfide bond.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1 and PL2 are peptide linkers
  • M is a masking moiety that binds to p40 subunit and inhibits a biological activity of IL-12
  • A is an IL-12 p35 subunit
  • B is an IL-12 p40 subunit
  • SS is a disulfide bond.
  • the non-cleavable peptide linker comprises an amino acid sequence from SEQ ID NOs: 29-33; and wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 34-54.
  • the prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 20; and wherein the second polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 21-28.
  • the prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 55; and wherein the second polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 56-59.
  • the prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 60; and the second polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 61-64.
  • the prodrug wherein the prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 65; and the second polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 66-73.
  • the prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 20; and the second polypeptide chain comprises an amino acid sequence at least 99% identical to one selected from SEQ ID NOs: 76-79.
  • the present disclosure provides a pharmaceutical composition comprising the present prodrugs and a pharmaceutically acceptable excipient.
  • the present disclosure provides a polynucleotide or polynucleotides encoding the present prodrugs, expression vectors comprising the polynucleotides, and host cells (e.g., mammalian host cells such as CHO, NS0 cells, and 293T cells) comprising the expression vectors.
  • a host cell described herein has the gene or genes encoding uPA, MMP-2, MMP-9 and/or matriptase knocked out.
  • the present disclosure also provides methods of making the present prodrugs, comprising culturing the host cells under conditions that allow expression of prodrugs and isolating (also purifying) the prodrugs. [0032]
  • the present disclosure also provides a method of treating a cancer or an infectious disease or stimulating the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the prodrug or the pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a method of treating cancer comprising administering to a cancer patient the prodrug or pharmaceutical composition of the present disclosure in combination with a second pharmaceutical composition, wherein the second pharmaceutical composition comprises an active ingredient selected from a cytokine other than IL-12 or its fusion molecule, an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-2 fusion molecule, a PD-1-IL-7 fusion molecule, a PD-1 antibody-IL-15 fusion molecule, and a PD-1-IL-21 fusion molecule.
  • a cytokine other than IL-12 or its fusion molecule an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-2 fusion molecule, a PD-1-IL-7 fusion molecule, a PD-1 antibody-IL-15 fusion molecule, and a PD-1-IL-21 fusion molecule
  • the patient may have, for example, a viral infection (e.g., HIV, HBV, HCV, or HPV infection), or a cancer selected from the group consisting of brain cancer, breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer.
  • a viral infection e.g., HIV, HBV, HCV, or HPV infection
  • a cancer selected from the group consisting of brain cancer, breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer.
  • prodrugs for use in treating a cancer or an infectious disease or stimulating the immune system in the present method; use of an Il-12 prodrug for the manufacture of a medicament for treating a cancer or an infectious disease or stimulating the immune system in the present method; and articles of manufacture (e.g., kits) comprising one or more dosing units of the present Il-12 prodrug.
  • FIGs.1A and 1B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a first masking moiety (MM1; white box), a second masking moiety (MM2; black box), a carrier (C), and two cleavable peptide linkers.
  • FIG.1A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of p35 through a non-cleavable peptide linker.
  • the second masking moiety is fused to the C-terminus of the other Fc domain polypeptide through a cleavable linker and the first masking moiety is fused to the C- terminus of the second masking moiety through a cleavable peptide linker.
  • FIG.1B shows the IL-12 subunit p40 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p35 is fused to the C-terminus of p40 through a non-cleavable peptide linker.
  • FIGs.2A and 2B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a first masking moiety (MM1; white box), a second masking moiety (MM2; black box), a carrier (C), and one cleavable peptide linker.
  • FIG.2A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of p35 through a non- cleavable peptide linker.
  • the second masking moiety is fused to the C-terminus of the other Fc domain polypeptide through a non-cleavable linker and the first masking moiety is fused to the C-terminus of the second masking moiety through cleavable peptide linker.
  • FIG.2B shows the IL-12 subunit p40 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p35 is fused to the C-terminus of p40 through a non-cleavable peptide linker.
  • the first masking moiety is fused to the C-terminus of the other Fc domain polypeptide through a non- cleavable linker and said second masking moiety is fused to the C-terminus of the first masking moiety through cleavable peptide linker.
  • FIGs.3A and 3B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a first masking moiety (MM1; white box), a second masking moiety (MM2; black box), a carrier (C), and two cleavable peptide linkers.
  • FIG.3A shows an IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while an IL-12 subunit p40 is fused to the C-terminus of the other Fc domain polypeptide, both through a non-cleavable peptide linker.
  • FIG.3B shows an IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of the other Fc domain polypeptide, both through a non-cleavable peptide linker.
  • FIGs.4A and 4B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a first masking moiety (MM1; white box), a second masking moiety (MM2; black box), a carrier (C), and one cleavable peptide linker.
  • FIG.4A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of the other Fc domain polypeptide, both through a non-cleavable peptide linker.
  • the second masking moiety is fused to the C-terminus of p35 through a cleavable linker and the first masking moiety is fused to the C-terminus of p40 through non-cleavable peptide linker.
  • FIG.4B shows an IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of the other Fc domain polypeptide, both through a non-cleavable peptide linker.
  • the second masking moiety is fused to the C-terminus of p40 through a cleavable linker and the first masking moiety is fused to the C-terminus of p35 through non- cleavable peptide linker.
  • FIGs.5A and 5B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a masking moiety (MM), and one cleavable peptide linker.
  • FIG.5A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of p35, both through a non-cleavable peptide linker.
  • the masking moiety is fused to the C- terminus of the second Fc through a cleavable linker.
  • FIG.5B shows the IL-12 subunit p40 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p35 is fused to the C-terminus of p40, both through a non-cleavable peptide linker.
  • the masking moiety is fused to the C-terminus of the second Fc through a cleavable linker.
  • FIGs.6A and 6B show an illustration of the structure of an IL-12 prodrug comprising an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a masking moiety (MM), a carrier (C), and no cleavable peptide linker.
  • FIG.6A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p40 is fused to the C-terminus of the other Fc domain polypeptide, both through a non-cleavable peptide linker.
  • the masking moiety is fused to the C-terminus of p35 through a non-cleavable linker.
  • FIG.6B shows the IL-12 subunit p40 fused to the C-terminus of one of the Fc domain polypeptides, while the IL-12 subunit p35 is fused to the C-terminus of p40, both through a non-cleavable peptide linker.
  • FIGs.7A and 7B show an illustration of the structure of an IL-12 prodrug comprises three polypeptide chains, which comprises an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a masking moiety (MM), and a carrier (C).
  • FIG.7A shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides, the masking moiety is fused to the C- terminus of the other Fc domain polypeptide, both optionally through a non-cleavable linker.
  • the p40 is the third polypeptide chain, which is linked to the p35 subunit through a disulfide bond.
  • FIG.7B shows the IL-12 subunit p35 fused to the C-terminus of one of the Fc domain polypeptides optionally through a non-cleavable peptide linker, the masking moiety is fused to the C-terminus of the other Fc domain polypeptide through a cleavable peptide linker.
  • the p40 is the third polypeptide chain, which is linked to the p35 subunit through a disulfide bond.
  • FIGs.8A and 8B show an SDS-PAGE analysis.
  • FIG.8A shows the SDS-PAGE analysis of the Protein A Affinity chromatography pool samples of the IL-12 prodrugs JR3.105.2, JR3.105.3, JR3.105.4, and JR3.105.5 with structures as illustrated in FIG.5B and sequences as shown in Table 2. The samples were run under reduced and non-reduced conditions. The samples were of reasonable purity though some levels of the activated versions of the prodrugs are visible.
  • FIG.8B shows the SDS-PAGE analysis of the prodrug JR3.105.5 sample prior to and after protease treatment in order to activate the prodrug. The data showed that the majority of the prodrug sample were activated.
  • FIG.9 shows a cell-based activity analysis of the IL-12 prodrug samples prior to and after protease treatment.
  • the prodrugs treated with protease are labeled as JR3.105.2-Clv, JR3.105.3-Clv, JR3.105.4-Clv, and JR3.105.5-Clv.
  • Significant activation was observed with the Prodrug JR3.105.5 after the treatment with protease.
  • FIG.10 shows the EC50 values of IL-12 prodrug samples tested in the cell-based assay, as shown in FIG.9.
  • the EC50 value for the Prodrug JR3.105.5 was approximately 0.9 nM prior to activation and approximately 0.05 nM after activation.
  • antigen-binding moiety refers to a polypeptide or a set of interacting polypeptides that specifically bind to an antigen, and includes, but is not limited to, an antibody (e.g., a monoclonal antibody, polyclonal antibody, a multi-specific antibody, a dual specific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody) or an antigen- binding fragment thereof (e.g., a Fab, a Fab’, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), or a diabody), a single chain antibody, and an Fc-containing polypeptide such as an immunoadhesin.
  • an antibody e.g., a monoclonal antibody, polyclonal antibody, a multi-specific antibody, a dual specific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody
  • the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG4).
  • the antibody may be of any light chain isotype (e.g., kappa or lambda).
  • the antibody may be human, non-human (e.g., from mouse, rat, rabbit, goat, or another non-human animal), chimeric (e.g., with a non-human variable region and a human constant region), or humanized (e.g., with non-human CDRs and human framework and constant regions).
  • the antibody is a derivatized antibody.
  • cytokine agonist polypeptide or “cytokine moiety” refers to a wildtype cytokine, or an analog thereof.
  • An analog of a wildtype cytokine has the same biological specificity (e.g., binding to the same receptor(s) and activating the same target cells) as the wildtype cytokine, although the activity level of the analog may be different from that of the wildtype cytokine.
  • the analog may be, for example, a mutein (i.e., mutated polypeptide) of the wildtype cytokine, and may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wildtype cytokine.
  • cytokine mask or “masking moiety” refers to a moiety (e.g., a polypeptide) that binds to a cytokine, thereby inhibiting the cytokine from binding to its receptor on the surface of a target cell and/or exerting its biological functions while being bound by the mask.
  • cytokine mask examples include, without limitations, a polypeptide derived from an extracellular domain of the cytokine’s natural receptor that makes contact with the cytokine.
  • effective amount or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease, such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
  • functional analog refers to a molecule that has the same biological specificity (e.g., binding to the same ligand) and/or activity (e.g., activating or inhibiting a target cell) as a reference molecule.
  • fused refers to the joining of the two polypeptide sequences through a backbone peptide bond.
  • Two polypeptides may be fused directly or through a peptide linker that is one or more amino acids long.
  • a fusion polypeptide may be made by recombinant technology from a coding sequence containing the respective coding sequences for the two fusion partners, with or without a coding sequence for a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.
  • composition when used to refer to an ingredient in a composition means that the excipient is suitable for administration to a treatment subject, including a human subject, without undue deleterious side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).
  • subject refers to a mammal and includes, but is not limited to, a human, a pet (e.g., a canine or a feline), a farm animal (e.g., cattle or horse), a rodent, or a primate.
  • treatment or “treating” is an approach for obtaining beneficial or desired clinical results.
  • Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, ameliorating a disease state, stabilizing a disease (e.g., preventing or delaying the worsening or progression of the disease), preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, providing partial or total remission of a disease, decreasing the dose of one or more other medications required to treat a disease, increasing the patient’s quality of life, and/or prolonging survival.
  • the methods of the present disclosure contemplate any one or more of these aspects of treatment.
  • IL-12 Prodrugs [0055] The present disclosure provides IL-12 prodrugs that are metabolized in vivo to become active IL-12 therapeutics.
  • the IL-12 prodrugs have fewer side effects, better in vivo PK profiles (e.g., longer half-life) and better target specificity, and are more efficacious as compared to prior IL-12 therapeutics.
  • the present IL-12 prodrugs comprise an IL-12 agonist polypeptide, at least one masking moiety (M), a carrier (C), and one or more cleavable or non-cleavable peptide linkers that link the masking moiety to the agonist polypeptide or the carrier.
  • the IL-12 prodrugs comprise an IL-12 agonist polypeptide, a first masking moiety (MM1), a second masking moiety (MM2), a carrier (C), and one or more cleavable or non- cleavable peptide linkers that link the masking moieties to the agonist polypeptide or the carrier.
  • IL-12 comprises subunits p40 and p35.
  • Subunit p40 comprises an amino acid as shown in SEQ ID NO: 5 and p35 comprises an amino acid sequence as shown in SEQ ID NO: 6.
  • an IL-12 analog comprises subunit p40 with an amino acid that is at least 90% identical to SEQ ID NO: 5.
  • an IL-12 analog comprises subunit p35 with an amino acid sequence that is at least 90% identical to SEQ ID NO: 6.
  • the IL-12 prodrugs comprise at least one masking moiety.
  • the masking moieties may be linked to the cytokine moiety or to the carrier moiety through a peptide linker.
  • the peptide linker is a cleavable peptide linker.
  • the cleavable peptide linker comprises one or more cleavable moieties, which are substrates of proteases typically found at a tumor site.
  • the mask inhibits the cytokine moiety’s biological functions while the mask is binding to it.
  • the mask inhibits a biological activity of IL-12 or its analog.
  • the prodrugs may be activated at a target site (e.g., at a tumor site or the surrounding environment) in the patient by cleavage of the linker and the consequent release of the cytokine mask from the prodrug, exposing the previously masked cytokine moiety and allowing the cytokine moiety to bind to its receptor on a target cell and exert its biological functions on the target cell.
  • the carriers for the IL-12 prodrugs are antigen-binding moieties that bind an antigen at a target site (e.g., tumor surface).
  • the present IL-12 prodrugs are metabolized to become active in the body at a target site targeted by the carrier.
  • the carrier in the prodrug is an antibody targeting a tumor antigen such that the IL-12 prodrug is delivered to a tumor site in a patient and is metabolized locally (e.g., inside or in the vicinity of the tumor microenvironment) through cleavage of a linker linking the cytokine mask to the carrier or the cytokine moiety, making the cytokine moiety available to interact with its receptor on a target cell and stimulating the target immune cells locally.
  • the carrier is selected from an albumin, a Fc fragment, a polyethylene glycol (PEG), or an antibody or antigen-binding fragment thereof.
  • the masking moiety for IL-12 from an IL-12 receptor beta1 subunit extracellular domain or a fragment thereof, an IL-12 receptor beta2 subunit extracellular domain or a fragment thereof, and a scFv or Fab with specificity to p40 or p35.
  • the masking moiety inhibits a biological activity of IL-12 or its analog.
  • the masking moiety comprises a scFv, wherein the scFv has an amino acid sequence at least 99% identical to SEQ ID NOs: 7-11 and 12.
  • the masking moiety is an IL-12 receptor extracellular domain (ECD) or its functional analog, wherein the IL-12 receptor is IL-12 receptor beta1.
  • the masking moiety is IL-12 receptor extracellular domain (ECD), its functional analog, or a fragment thereof, wherein the IL-12 receptor is IL-12 receptor beta1.
  • IL-12 receptor beta1 has an amino acid sequence that is at least 95% identical to SEQ ID NO: 137.
  • the prodrug comprises two masking moieties MM1 and MM2; wherein at least one of the masking moieties comprises a scFv or Fab, which binds to p40 or p35, and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1 or IL-12R ⁇ 2.
  • the scFv or Fab comprises the same light chain CDRs and heavy chain CDRs as IL-12 antibody briakinumab or ustekinumab.
  • the scFv or Fab comprises the same light chain CDRs and heavy chain CDRs as an IL-12 antibody selected from PMA204, Antibody 1, Antibody 50, Antibody 68, Antibody 80, and Antibody 136 (see U.S. Patent No.8,563,697).
  • both masking moieties are scFvs or Fabs; wherein the first masking moiety binds to p40 and reduces or inhibits the binding of IL-12 to IL- 12R ⁇ 1, and the second masking moiety binds to p40 or p35, and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2.
  • the first masking moiety comprises the same light chain CDRs and heavy chain CDRs as an IL-12 antibody briakinumab or ustekinumab.
  • the second masking moiety comprises the same light chain CDRs and heavy chain CDRs as an IL-12 antibody selected from PMA204, Antibody 1, Antibody 50, Antibody 68, Antibody 80, and Antibody 136 (see U.S. Patent No.8,563,697).
  • the carrier moieties of the present prodrugs may be an antigen-binding moiety, or a moiety that is not antigen-binding.
  • Non-antigen-binding carrier moieties may be used for the present prodrugs.
  • an antibody Fc domain e.g., a human IgG 1 , IgG 2 , IgG 3 , or IgG 4 Fc
  • a polymer e.g., PEG
  • an albumin e.g., a human albumin
  • a nanoparticle can be used.
  • the carrier moiety of the prodrug may comprise an albumin (e.g., human serum albumin) or a fragment thereof.
  • the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human serum albumin or a fragment thereof.
  • the carrier moiety comprises an albumin fragment (e.g., a human serum albumin fragment) that is about 10 or more, 20 or more, 30 or more 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length.
  • an albumin fragment e.g., a human serum albumin fragment
  • the albumin fragment is between about 10 amino acids and about 584 amino acids in length (such as between about 10 and about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids in length).
  • the albumin fragment includes the Sudlow I domain or a fragment thereof, or the Sudlow II domain or the fragment [0066]
  • the carrier is an antibody Fc fragment. Fc is a dimeric molecule that has two N-terminals and two C-terminals.
  • the cytokine moiety can be fused to one Fc polypeptide in a dimeric Fc fragment, and the masking moieties can be fused to the 2 nd Fc polypeptide.
  • both the cytokine moiety and the masking moiety are fused to the C-terminal of each polypeptide chain of the dimeric Fc fragment.
  • both the cytokine moiety and the masking moieties are fused to the N-terminal of each polypeptide chain of the dimeric Fc fragment. In either case, at least one of the masking moieties is fused to the Fc polypeptide directly or indirectly through a cleavable peptide linker.
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-B, F2-CL1-MM2-CL2-MM1 (FIG.1A); and b.
  • F1-PL1-B-PL2-A, F2-CL1-MM1-CL2-MM2 (FIG.1B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; CL1 and CL2 are cleavable peptide linkers; MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1; MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1 and PL2 are peptide link
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-B, F2-PL3-MM2-CL-MM1 (FIG.2A); and b.
  • F1-PL1-B-PL2-A, F2-PL3-MM1-CL-MM2 (FIG.2B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1, PL2, and PL3 are peptide linkers; CL is a cleavable peptide linker; MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL- 12R ⁇ 1; MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1, PL2, and PL3
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-CL1-MM2, F2-PL2-B-CL2-MM1 (FIG.3A); and b.
  • F1-PL1-A-CL1-MM1, F2-PL2-B-CL2-MM2 (FIG.3B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; CL1 and CL2 are cleavable peptide linkers; MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1; MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers;
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-CL-MM2, F2-PL2-B-PL3-MM1 (FIG.4A); and b.
  • F1-PL1-A-PL2-MM1, F2-PL3-B-CL-MM2 (FIG.4B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1, PL2, and PL3 are peptide linkers; CL is a cleavable peptide linker; MM1 is the first masking moiety, which binds to p40 and reduces or inhibits the binding of IL-12 to IL- 12R ⁇ 1; MM2 is the second masking moiety, which binds to p35 and/or p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 2; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1, PL2, and PL3 are
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-B, F2-CL-M (FIG.5A); and b.
  • F1-PL1-B-PL2-A, F2-CL-M (FIG.5B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; CL is a cleavable peptide linker; M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1 and PL2 are peptide linkers
  • CL is a cleavable peptide linker
  • M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1
  • A
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-A-PL2-M, F2-PL3-B (FIG.6A); and b.
  • F1-PL1-B-PL2-A, F2-PL3-M (FIG.6B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1, PL2, and PL3 are peptide linkers; M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1, PL2, and PL3 are peptide linkers
  • M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1
  • A is an IL-12 p35 subunit
  • B is an IL-12 p40 subunit.
  • the carrier moiety is an Fc domain comprising a first and a second polypeptide chain (i.e., two different heavy chains), wherein the polypeptide chains of the prodrug comprise molecular formulae (from N-terminus to C-terminus) selected from one of the following pairs: a. F1-PL1-M, F2-PL2-A-SS-B (FIG.7A); and b.
  • F1-CL-M F2-PL1-A-SS-B (FIG.7B); wherein F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer; PL1 and PL2 are peptide linkers; M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1; CL is a cleavable peptide linker; A is an IL-12 p35 subunit; and B is an IL-12 p40 subunit; and SS is a disulfide bond. 2.
  • F1 and F2 are subunits of the carrier moiety (e.g., Fc domain), which form a heterodimer
  • PL1 and PL2 are peptide linkers
  • M is a masking moiety that binds to p40 and reduces or inhibits the binding of IL-12 to IL-12R ⁇ 1
  • CL is a
  • the carrier moiety may be an antibody or an antigen-binding fragment thereof, or an immunoadhesin.
  • the antigen-binding moiety is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab’ fragment, a F(ab’) 2 fragment, a Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single- chain variable fragment (scFv).
  • the antigen-binding moiety is a bispecific antigen-binding moiety and can bind to two different antigens or two different epitopes on the same antigen.
  • the antigen-binding moiety may provide additional and potentially synergetic therapeutic efficacy to the cytokine agonist polypeptide.
  • the antigen- binding moiety comprises a full-length antibody heavy chain or a full-length antibody light chain.
  • the antigen-binding moiety includes an antibody heavy chain fragment or an antibody light chain fragment.
  • the cytokine moiety is fused to the C-terminus of one of the heavy chains of an antibody, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a peptide linker (optionally a cleavable linker), wherein the two heavy chains optionally contain mutations that allow the specific pairing of the two different heavy chains.
  • a peptide linker optionally a cleavable linker
  • the two heavy chain polypeptides in the prodrug may form stable heterodimers through “knobs-into- holes” mutations.
  • “Knobs-into-holes” mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies (see, e.g., U.S. Pat.8,642,745).
  • the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the “knob chain” and T366S, L368A, and/or Y407V mutations in the CH3 domain of the “hole chain.”
  • An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the “knobs chain” and an E356C or S354C mutation into the CH3 domain of the “hole chain” (see, e.g., Merchant et al., Nature Biotech. (1998) 16:677-81).
  • the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain.
  • the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge (numbering always according to EU index of Kabat; Kabat et al., “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • knobs-into-holes technologies can be used alternatively or additionally.
  • another example of knobs-into-holes mutations for an antibody moiety is having R409D/K370E mutations in the CH3 domain of the “knob chain” and D399K/E357K mutations in the CH3 domain of the “hole chain” (EU numbering).
  • the antibody moiety in the prodrug comprises L234A and L235A (“LALA”) mutations in its Fc domain.
  • LALA mutations eliminate complement binding and fixation as well as Fc ⁇ dependent ADCC (see, e.g., Hezareh et al. J. Virol.
  • the LALA mutations are present in the antibody moiety in addition to the knobs-into-holes mutations.
  • the antibody moiety comprises the M252Y/S254T/T256E (“YTE”) mutations in the Fc domain.
  • YTE M252Y/S254T/T256E
  • the YTE mutations allow the simultaneous modulation of serum half-life, tissue distribution and activity of IgG 1 (see Dall’Acqua et al., J Biol Chem. (2006) 281: 23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147- 53).
  • the YTE mutations are present in the antibody moiety in addition to the knobs-into-holes mutations.
  • the antibody moiety has YTE, LALA and knobs-into-holes mutations or any combination thereof.
  • the antibody binds to PD-L1.
  • the antibody binds to CEA.
  • the antibody binds to an antigen on a tumor cell, for examples, 5T4, FAP, Trop-2, PD-L1, HER-2, EGFR, Claudin 18.2, DLL-3, GCP3, and CEA.
  • the antibody may or may not have Antibody-Dependent Cellular Cytotoxicity (ADCC) activity.
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • the antibody may also be further conjugated with cytotoxic drugs.
  • the antibody binds to a target on the surface of an immune cell and has the ability to activate said immune cell and enhance its anti-cancer activity, for examples, PD-1 antibody, LAG3 antibody, TIGIT antibody, TGF-beta antibody, and CTLA4 antibody.
  • the antigen-binding moiety can bind an antigen on the surface of a cell, such as a cancer cell.
  • the antigen-binding moiety is a bispecific antigen-binging moiety, can bind to two different antigens or two different epitopes on the same antigen.
  • the antigen-binding moiety binds to Guanyl cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen (CEA), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta- like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1) 1, vascular endothelial growth factor receptor 2 (VEGFR2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), Thomsen-Frieden
  • GCC
  • the antigen-binding moiety binds to an epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen- binding moiety binds to a Delta/Serrate/Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after the 374 th amino acid of GPC3. In some embodiments, the antigen-binding moiety binds to a heparin sulfate glycan of GPC3. In some embodiments, the antigen-binding moiety binds to Claudin 18.2 and does not bind to Claudin 18.1.
  • the antigen-binding moiety binds to Claudin 18.1 with at least 10 times weaker binding affinity than to Claudin 18.2.
  • the antigen-binding moiety can bind an antigen on the surface of a cell, such as an immune cell, for example T cells, NK cells, and macrophages.
  • the antigen-binding moiety is a bispecific antigen-binging moiety, can bind to two different antigens or two different epitopes on the same antigen.
  • the antigen-binding moiety binds to PD-1, LAG-3, TIM-3, CTLA-4, or TGF-beta.
  • the antigen-binding moiety includes an antibody or fragment thereof known in the art that binds to PD-1 and disrupts the interaction between the PD-1 and its ligand (PD-L1) to stimulate an anti-tumor immune response.
  • the antibody or antigen-binding portion thereof binds specifically to PD-1.
  • antibodies that target PD-1 and which can find use in the present invention include, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti- PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene), and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis).
  • the PD-1 antibody is from clone: RMP1-14 (rat IgG)—BioXcell cat# BP0146.
  • Other suitable anti-PD-1 antibodies include those disclosed in U.S. Pat. No.8,008,449.
  • the antibody or antigen-binding portion thereof binds specifically to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibodies known in the art which bind to PD-L1 and disrupt the interaction between the PD-1 and PD-L1, and stimulates an anti-tumor immune response, are suitable for use in combination treatment methods disclosed herein.
  • antibodies that target PD-L1 include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech; currently in human trials).
  • BMS-936559 Bristol-Myers Squibb
  • MPDL3280A Genetech; currently in human trials
  • Other suitable antibodies that target PD-L1 are disclosed in U.S. Pat. No.7,943,743. It will be understood by one of ordinary skill that any antibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response, is suitable for use in the combination treatment methods disclosed herein.
  • the carrier is an antibody against human PD-L1 is selected from ASKB1296, avelumab, atezolizumab and durvalumab.
  • antigen-binding moieties include trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-FAP-alpha antibody sibrotuzumab (BIBH1), and fragments thereof.
  • the antigen-binding moiety that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), sibrotuzumab (BIBH1), or a fragment thereof.
  • the antigen-binding moiety has an antibody heavy chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), sibrotuzumab (BIBH1), or a fragment thereof.
  • the antigen-binding moiety has an antibody light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), sibrotuzumab (BIBH1), or a fragment thereof.
  • the antigen-binding moiety comprises the six complementarity-determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806, or sibrotuzumab (BIBH1).
  • CDRs complementarity-determining regions
  • BIBH1 anti-EGFR antibody mAb806, or sibrotuzumab
  • the “Kabat” Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). “Chothia” CDRs refer to the location of the structural loops (Chothia & Lesk, J. Mol. Biol. (1987) 196:901-917).
  • the “AbM” CDRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.
  • the “Contact” CDRs are based on an analysis of the available complex crystal structures.
  • amino acid number of antibodies refers to the Kabat numbering scheme as described in Kabat et al., supra, including when CDR delineations are made in reference to Kabat, Chothia, AbM, or Contact schemes.
  • the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a framework region (FR) or CDR of the variable domain.
  • FR framework region
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.
  • the CDRs are “extended CDRs,” and encompass a region that begins or terminates according to a different scheme.
  • an extended CDR can be as follows: L24—L36, L26—L34, or L26—L36 (VL-CDR1); L46—L52, L46—L56, or L50—L55 (VL-CDR2); L91—L97 (VL-CDR3); H47—H55, H47—H65, H50—H55, H53—H58, or H53— H65 (VH-CDR2); and/or H93—H102 (VH-CDR3).
  • the IL-12 prodrug of the present disclosure comprises a carrier comprising an antigen-binding moiety; wherein the antigen-binding moiety binds to Trop-2.
  • the IL-12 prodrug is used to treat patients with solid tumors. In some embodiments, the prodrug is used to treat triple negative breath cancer, urothelial cancer, small- cell lung cancer, pancreatic cancer, hilar cholangiocarcinoma, cervical cancer, and gastric cancer. [0088] In some embodiment, the IL-12 prodrug comprises a carrier comprising an antigen- binding moiety; wherein the antigen-binding moiety binds to 5T4. In some embodiments, the IL-12 prodrug is used to treat patients with solid tumor. In some embodiments, the prodrug is used to treat triple negative breath cancer, small-cell lung cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, and gastric cancer.
  • the IL-12 prodrug comprises a carrier comprising an antigen- binding moiety; wherein said antigen-binding moiety binds to Claudin 18.2. In some embodiments, the IL-12 prodrug is used to treat patients with pancreatic cancer and gastric cancer. [0090] In some embodiments, the IL-12 prodrug comprises a carrier comprising an antigen- binding moiety; wherein said antigen-binding moiety binds to EGFR Type III. In some embodiment, said IL-12 prodrug is used to treat patients with glioblastoma and colon cancer.
  • the IL-12 prodrugs are used in combination with an immune checkpoint blockade, such as a PD-1 antibody or an PD-1 antibody fragment thereof.
  • an immune checkpoint blockade such as a PD-1 antibody or an PD-1 antibody fragment thereof.
  • C. Linker Components of the Prodrugs [0092] The IL-12 agonist polypeptide may be fused to the carrier moiety with or without a peptide linker. The peptide linker may be noncleavable. In some embodiments, the peptide linker is selected from SEQ ID NOs: 29-33. [0093] The IL-12 mask may be fused to the cytokine moiety, to the carrier, or to another mask through a noncleavable linker or a cleavable linker.
  • the cleavable linker may contain one or more (e.g., two or three) cleavable moieties (CM).
  • CM cleavable moieties
  • Each CM may be a substrate for an enzyme or protease selected from legumain, plasmin, TMPRSS-3/4, MMP2, MMP9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, and PSA.
  • cleavable linkers include, without limitation, those comprising an amino acid sequence selected from SEQ ID NOs: 34-54.
  • the IL-12 prodrug of the present disclosure further comprises another cytokine or effector moiety.
  • the second cytokine moiety comprises an IL-2 agonist polypeptide.
  • the IL-2 agonist polypeptide in the prodrug comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:1.
  • the IL-2 agonist polypeptide contains mutations which lead to the significantly reduced affinity to the high-affinity trimeric IL-2 receptor.
  • the high-affinity trimeric IL-2 receptor comprises alpha, beta, and gamma IL-2 receptor subunits.
  • the IL-2 agonist polypeptide further comprises mutations that lead to significantly reduced binding affinity to the moderate-affinity IL-2 receptor, which comprises the IL-2 receptor beta and gamma subunits.
  • the IL-2 agonist polypeptide comprises one or more mutations at residues R38, F42, F44, Y45, E62, E68, L72, and A73 according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the IL-2 agonist polypeptide further comprises one or more mutations at residues D20, N88, N90, and Q126 according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the IL-2 agonist polypeptide of the IL-2 prodrug comprises mutations at residues F42N/F44T, A73T, and/or N90T; wherein the mutations are referred to according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the IL-2 agonist polypeptide of the IL-2 prodrug comprises mutations selected from one of the following combinations: T3A, R38A, F42I, Y45N, E62L, E68V, C125S; T3A, R38K, F42K, Y45R, E62L, E68V, C125S; T3A, R38A, F42A, Y45A, E62A, C125S; T3A, R38S, F42A, Y45A, E62A, C125S; or R38S, F42A, Y45A, E62A; and wherein said mutations are referred to according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the IL-2 agonist polypeptide is at least 30 times lower affinity in binding to the high-affinity trimeric IL-2 receptor comprising the alpha, beta, and gamma subunits as compared to the wild type IL-2; wherein the polypeptide comprises one or more mutations at R38, F42, Y45, E62, E68, and L72; wherein the mutations are referred to according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the agonist polypeptide of IL-2 further comprises one or more mutations at residues A3, D20, N88, C125 and/or Q126 according to the numbering of the human IL-2 with amino acid sequence of SEQ ID NO: 1.
  • the IL-2 agonist peptide comprises an amino acid sequence selected from SEQ ID NOs: 74 and 75. 2.
  • IL-15 Agonist Polypeptides [00100]
  • the IL-12 prodrug of the present disclosure further comprises another cytokine or effector moiety.
  • the second cytokine moiety comprises an IL-15 agonist polypeptide.
  • the IL-15 agonist polypeptide may optionally comprise the Sushi domain of the IL-15 receptor alpha subunit.
  • the IL-15 moiety is an IL-15 mutein comprising at least 1, 2, 3, 4, or 5 mutations at positions selected from N1, N4, I6, S7, D8, K10, K11, E46, D61, T62, E64, N65, I68, L69, N72, V63, L66, I67, A70, N71, Q108, N112 of human IL-15.
  • Exemplary IL-15 muteins are those with one or more mutations selected from N1A, N1D, N4A, N4D, I6T, S7A, D8A, DAT, D8E, D8N, K10A, K10D, K11A, K11D, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I 67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I67E, I67D, Q108E, and N112A.
  • the IL-15 moiety comprises a mutation at positions selected from E46, V49, L45, S51, and L52. Unless otherwise indicated, all residue numbers in IL-15 and IL-15 muteins described herein are in accordance with the numbering in SEQ ID NO: 4. In other embodiments, the IL-15 moiety comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.
  • the IL-15 mutein contains mutations selected from N1D/D61N, N1D/E64Q, N4D/D61N, N4D/E64Q, D8N/D61N, D8N/E64Q, D30N/E64Q/N65D, D61N/E64Q, E64Q/Q108E, N1D/N4D/D8N, D61N/E64Q/N65D, N1D/D61N/E64Q, N1D/D61N/E64Q/Q108E, and N4D/D61N/E64Q/Q108E.
  • the present IL-15 prodrug comprises an IL-15R ⁇ Sushi domain.
  • the Sushi domain may be fused to the carrier directly or to the IL-15 cytokine moiety, optionally through a linker (e.g., a noncleavable or cleavable peptide linker).
  • the masking moiety may be fused to the Sushi domain or to the carrier through a cleavable or noncleavable peptide linker.
  • the Sushi domain is fused to the carrier and the cytokine moiety is fused to the Sushi domain through a peptide linker. 3.
  • masking moieties of the additional cytokine moieties disclosed herein comprise a peptide, monomer, or dimer of a soluble extracellular domain of an IL-2 receptor beta subunit, a soluble extracellular domain of IL-2 receptor beta and gamma subunit fusion protein or complex, or antibodies that bind to IL-2 or IL-15 and interfere with the binding of IL-2, IL-2 muteins, or IL-15 to the dimeric IL-2 receptor.
  • an IL-2 masking moiety also inhibits or reduces the biological activities of IL-2 or IL-15.
  • the IL-2 masking moiety is a peptide identified from a peptide library through screening. In some embodiments, the IL-2 masking moiety is an antibody or fragment thereof that blocks the binding of IL-2 or IL-2 mutants to an IL-2 receptor. In some embodiments, the IL-2 masking moiety is an scFv, a Fab or a single chain Fab having the same CDR domain sequences as the antibody selected from hybridoma clones 4E12B2D10, 4E12B2, and 4E12. II.
  • the IL-12 prodrug comprises an antibody or an Fc domain, and an IL-12 agonist polypeptide; wherein the IL-12 agonist polypeptide is fused to the C-terminal of one of the heavy chains of the antibody or Fc polypeptide chain of the Fc domain, optionally through a peptide linker; wherein said IL-12 masking moiety is fused to the C-terminal of the 2 nd heavy chain or Fc polypeptide chain, through a cleavable peptide linker.
  • prodrugs examples are illustrated in FIGs.1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 6A, 6B, 7A, and 7B. All the prodrugs are shown with the cytokine components p35 and p40 and the masking moieties located on the C-termini of the Fc domains. In some embodiments, the cytokine components p35 and p40 and the masking moieties are located on the N-termini of the Fc domains. In addition, prodrugs may comprise both IL-12 and IL-2 agonist polypeptides or both IL-12 and IL- 15 agonist polypeptides.
  • an IL-12 prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 20; and the second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 21-28.
  • the IL-12 prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 55; and said second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 56-59.
  • the IL-12 prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 60; and the second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 61-64.
  • the IL-12 prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 65; and the second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 66-73.
  • the IL-12 prodrug comprises two polypeptide chains, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 20; and the second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 76-79.
  • the second cytokine moiety is an IL-2 agonist polypeptide, such as one with an amino acid sequence selected from SEQ ID NOs: 74 and 75.
  • the second cytokine may be fused to the N-terminus of one of the Fc domain polypeptides, while the IL-12 agonist polypeptides and masking moieties are fused to the C-termini of the Fc domains, as illustrated in FIGs.1A-7B.
  • the IL-12 prodrugs may further comprise one or more masking moieties that bind to and inhibit a biological activity of the second cytokine.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl
  • Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.
  • Preservatives are added to retard microbial growth, and are typically present in a range from 0.2% - 1.0% (w/v).
  • Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3- pentanol, and m-cresol.
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride
  • benzalkonium halides e.g., chloride, bromide, iodide
  • Tonicity agents sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra- molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients.
  • Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Non-ionic surfactants or detergents also known as “wetting agents” are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
  • Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
  • Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride.
  • the choice of pharmaceutical carrier, excipient or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).
  • compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be administered by a number of routes.
  • said formulation is administrated directly in a tumor or tumors.
  • an antibody or protein formulation is a lyophilized formulation.
  • an antibody or protein formulation is an aqueous formulation.
  • the pharmaceutical composition is a combination pharmaceutical composition, which comprises an IL-12 prodrug of the present disclosure, a pharmaceutically acceptable excipient, and a second active ingredient selected from a cytokine other than IL-12 or its fusion molecule, an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-15 fusion molecule, a PD-1-IL-2 fusion molecule, and a PD-1-IL-21 fusion molecule.
  • a cytokine other than IL-12 or its fusion molecule an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-15 fusion molecule, a PD-1-IL-2 fusion molecule, and a PD-1-IL-21 fusion molecule.
  • the prodrugs disclosed herein are used to treat cancer.
  • the prodrugs are used to treat an infection, for example when the drug molecule is an antibacterial agent or an antiviral agent.
  • a method of treating a disease (such as cancer, a viral infection, or a bacterial infection) in a subject comprises administering to the subject an effective amount of the presently disclosed prodrugs.
  • the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer.
  • Exemplary cancers that may be treated include, but are not limited to, leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, stomach cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer.
  • the presently disclosed prodrugs are used to treat a bacterial infection such as sepsis.
  • the bacteria causing the bacterial infection are drug-resistant bacteria.
  • the antigen-binding moiety binds to a bacterial antigen.
  • the prodrug is used to treat a viral infection.
  • the virus causing the viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV), a human papilloma virus (HPV).
  • the antigen-binding moiety binds to a viral antigen.
  • dosages and routes of administration of the present pharmaceutical compositions are determined according to the size and condition of the subject, according to standard pharmaceutical practice.
  • the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, or intraspinally.
  • the composition is administered to a subject intravenously.
  • the prodrug is administered to a subject in need a single dose or a repeated dose.
  • the doses are given to a subject once per day, twice per day, three times per day, or four or more times per day. In some embodiments, about 1 or more (such as about 2, 3, 4, 5, 6, or 7 or more) doses are given in a week. In some embodiments, the antibody fusion molecule conjugated to the drug is administered weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, weekly for two weeks out of 3 weeks, or weekly for 3 weeks out of 4 weeks. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a course of treatment is about 1 or more doses (such as about 2, 2, 3, 4, 5, 7, 10, 15, or 20 or more doses).
  • the IL-12 prodrug is administered to a subject in combination with a second pharmaceutical composition, wherein the second pharmaceutical composition comprises an active ingredient selected from a cytokine other than IL-12 or its fusion molecule, an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-2 fusion molecule, a PD-1-IL-7 fusion molecule, a PD-1 antibody-IL-15 fusion molecule, and a PD-1-IL-21 fusion molecule.
  • the second pharmaceutical composition comprises an active ingredient selected from a cytokine other than IL-12 or its fusion molecule, an antibody against PD-1, an antibody against PD-L1, an antibody against CTLA-4, an antibody against CD47, a PD-1 antibody-IL-2 fusion molecule, a PD-1-IL-7 fusion molecule, a PD-1 antibody-IL-15 fusion molecule, and a PD-1-IL-21 fusion molecule.
  • Nucleic acid molecules encoding the polypeptide or the fusion polypeptide of said prodrug can be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid molecules may be readily isolated and sequenced using conventional methods.
  • Suitable host cells for cloning or expression of fusion polypeptide vectors include prokaryotic cells or eukaryotic cells. Exemplary host cells include Chinese Hamster Ovary (CHO) cells or human embryonic kidney cells (e.g., HEK293).
  • Expression host cells express the antibody fusion molecule. After an expression period, the host cells can by lysed and the prodrug or antibody fusion molecule can be purified.
  • Exemplary purification methods include liquid chromatography, such as ion exchange chromatography, affinity chromatography (such as Protein A affinity chromatography), or size exclusion chromatography.
  • a Prodrug of IL-12 which comprises an IL-12 p40 subunit (p40), an IL-12 p35 subunit (p35), a first masking moiety (MM), a second masking moiety, a carrier (C), and at least one cleavable peptide linker; wherein: a. Said IL-12 subunit p40 has an amino acid sequence at least 95% identical to that of SEQ ID NO: 5; b. Said IL-12 subunit p35 has an amino acid sequence at least 95% identical to that of SEQ ID NO: 6; c.
  • Said first masking moiety comprises a binding moiety which binds to p40, and said second masking moiety comprises a binding moiety which binds to p35; and d.
  • Said carrier is selected from an albumin or albumin fragment, an Fc domain, and an antibody.
  • said first masking moiety is selected from the extracellular domain of IL-12 receptor ⁇ 1 (IL-12R ⁇ 1 ECD) or a fragment thereof, a p40-binding peptide discovered from screening of a peptide library, and a single chain Fv or Fab domain of an antibody which binds to p40. 3.
  • said first masking moiety comprises the extracellular domain (ECD) of IL-12 receptor beta1 (IL-12R ⁇ 1) or a fragment thereof, wherein said IL-12R ⁇ 1 ECD comprises an amino acid sequence at least 95% identical as that of SEQ ID NO: 13. 5.
  • said first masking moiety comprises a fragment of IL-12R ⁇ 1 ECD, wherein said fragment comprises an amino acid sequence at least 95% identical as that of SEQ ID NO: 14, 15, or 16.
  • said second masking moiety comprises the extracellular domain (ECD) of IL-12 receptor beta2 (IL-12R ⁇ 2) or a fragment thereof, wherein said IL-12R ⁇ 2 ECD comprises an amino acid sequence at least 95% identical as that of SEQ ID NO: 17.
  • said second masking moiety comprises a fragment of IL-12R ⁇ 2 ECD, wherein said fragment comprises an amino acid sequence at least 95% identical as that of SEQ ID NOs: 18 or 19.
  • said first masking moiety comprises a scFv which binds to p40, wherein said scFv comprises an amino acid sequence at least 99% identical as one selected from SEQ ID NOs: 7-11 and 12.
  • said first masking moiety is selected from the IL-12R ⁇ 1 ECD or a fragment thereof comprising an amino acid sequence selected from SEQ ID NOs: 13-16, or a scFv with an amino acid sequence selected from SEQ ID NOs: 7-12; and wherein said second masking moiety comprises the IL-12R ⁇ 2 ECD or a fragment thereof comprising an amino acid sequence selected from SEQ ID NOs: 17-19.
  • the prodrug of any of embodiments 1-3 wherein said first masking moiety is selected from the IL-12R ⁇ 1 ECD or a fragment thereof comprising an amino acid sequence selected from SEQ ID NOs: 13-16, or a scFv with an amino acid sequence selected from SEQ ID NOs: 7-12; and wherein said second masking moiety comprises a scFv which binds to p35.
  • said carrier is an antibody which binds to a target expressed on the surface of an immune cell or a cancer cell.
  • cytokine moiety comprises an IL-2 agonist polypeptide, which comprises an amino acid sequence selected from SEQ ID NOs: 74 and 75.
  • said carrier comprises an Fc domain which comprises a first polypeptide chain F1 and a second polypeptide chain F2, wherein said F1 and F2 forms heterodimerization; wherein said p40 or its analog is fused to the C-terminus of F1 optionally through a non-cleavable peptide linker, said p35 or its analog is fused to the C- terminus of p40 through a non-cleavable peptide linker; and wherein said first masking moiety is fused to the C-terminus of F2 through a cleavable or non-cleavable peptide linker, and said second masking moiety is fused to the C-terminus of the said first masking moiety through a cleavable peptide link
  • said carrier comprises an Fc domain which comprises a first polypeptide chain F1 and a second polypeptide chain F2, wherein said F1 and F2 forms heterodimerization; wherein said p40 is fused to the C-terminus of F1 optionally through a non-cleavable peptide linker, and said first masking moiety is fused to the C-terminus of p40 through a cleavable or non-cleavable peptide linker; and wherein said p35 is fused to the C-terminus of F2, and said second masking moiety is fused to the C-terminus of p35 through a cleavable peptide linker. 16.
  • said carrier comprises an Fc domain which comprises a first polypeptide chain F1 and a second polypeptide chain F2, wherein said F1 and F2 forms heterodimerization; wherein said p35 is fused to the C-terminus of F1 optionally through a non-cleavable peptide linker, and said first masking moiety is fused to the C-terminus of p35 through a cleavable or non-cleavable peptide linker; and wherein said p40 is fused to the C-terminus of F2, and said second masking moiety is fused to the C-terminus of p40 through a cleavable peptide linker. 17.
  • said prodrug of embodiment 1 which comprises two polypeptide chains, wherein said first polypeptide chain comprises an amino acid sequence at least 99% identical as one selected from SEQ ID NO: 20; and said second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 21-28. 19.
  • the prodrug of embodiment 1 which comprises two polypeptide chains, wherein said first polypeptide chain comprises an amino acid sequence at least 99% identical as SEQ ID NO: 55; and said second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 56-59.
  • 20 The prodrug of embodiment 1 which comprises two polypeptide chains, wherein said first polypeptide chain comprises an amino acid sequence at least 99% identical as one selected from SEQ ID NO: 60; and said second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 61-64. 21.
  • the prodrug of embodiment 1 which comprises two polypeptide chains, wherein said first polypeptide chain comprises an amino acid sequence at least 99% identical as SEQ ID NO: 65; and said second polypeptide chain comprises an amino acid sequence at least 99% identical as on selected from SEQ ID NOs: 66-73. 22.
  • a pharmaceutical composition comprising the prodrug of any one of embodiments 1-21 and a pharmaceutically acceptable excipient.
  • 23. A polynucleotide or polynucleotides encoding the prodrug of any one of embodiments 1- 22.
  • 24. An expression vector or vectors comprising the polynucleotide or polynucleotides of embodiment 23.
  • a host cell comprising the vector(s) of embodiment 24. 26.
  • the host cell of embodiment 25, wherein the host cell has the gene or genes encoding uPA, MMP-2, MMP-9 and/or matriptase are knocked out.
  • 27. A method of making the prodrug of any one of embodiments 1-21, comprising culturing the host cell of claim 25 or 26 under conditions that allow expression of the antigen-binding molecule, and isolating the antigen-binding molecule.
  • 28. A method of treating a cancer or an infectious disease or stimulating the immune system in a patient in need thereof, comprising administering the pharmaceutical composition of embodiment 22.
  • invention 28 wherein said cancer is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer.
  • a method of treating of a patient with cancer comprising administering the pharmaceutical composition of claim 22 directly into a tumor or tumors.
  • Example 1 Transient Transfection of the IL-12 Prodrugs Using HEK293 Cells
  • Expression plasmids were co-transfected into approximately 4 x 10 6 cell/ml freestyle HEK293 cells at approximately 2.5 ⁇ g/ml using PEI (polyethylenimine).
  • PEI polyethylenimine
  • Fc-based IL-12 prodrugs the Fc-IL-12 fusion polypeptide and the Fc-masking moiety fusion polypeptide were in a 1:2 ratio.
  • the cell cultures were harvested 6 days after transfection by centrifuging at 9,000 rpm for 45 min followed by 0.22 ⁇ M filtration. A number of IL-12 prodrugs were expressed.
  • the Sequence ID NOs are listed in Table 2. Table 2.
  • Example 2 Purification of the Fc-Based IL-12 Prodrugs
  • the purifications of the proteins of the Fc-based IL-12 prodrugs were carried out by using Protein A Affinity. Briefly, the supernatant of the transient expression cell culture was loaded onto a Protein A column, which was equilibrated with 25 mM Tris-HCl, 30 mM NaCl, pH 7.8 (buffer A) before applying the sample. The column was washed with 5-column volumes of buffer A and the bound protein was eluted with 50 mM acetic acid, pH 3.6. The pH of the eluted protein was adjusted to approximately 5.2 using 1 M Tris base.
  • Example 3 SDS-PAGE Analysis
  • 10 ⁇ l of the 10-20 ⁇ g of purified protein samples were mixed with Bolt TM LDS Sample Buffer (Novex) with or without reducing reagents. The samples were heated at 70oC for 3 min and then loaded to a NuPAGE TM 4-12% BisTris Gel (InvitrogenTM). The gel was run in NuPAGE TM MOPS SDS Running buffer (InvitrogenTM) at 200 Volts for 40 min and then stained with Coomassie.
  • the purified samples of prodrugs were analyzed by the SDS-PAGE analysis, as shown in FIG.8A.
  • the data show that the masking moieties of the prodrugs were largely removed by the protease digestion, and that the activated molecules migrated at the expected molecular weights.
  • the Protein A column pool sample also contained activated version of the IL-12 prodrug (FIG.8B).
  • Example 4 Proteolytic Treatment
  • the proteases, human MMP2, human MMP9, mouse MMP2 and mouse MMP9 were purchased from R&D systems.
  • the protease digestion was carried out by incubating 10 ⁇ g-50 ⁇ g of prodrugs with 1 ⁇ g of human MMP2, human MMP9, mouse MMP2 or mouse MMP9 in the HBS buffer (20 mM HEPES, 150 mM NaCl2, pH 7.4) containing 2 mM CaCl2 and 10 ⁇ M ZnCl 2 at 37 o C for 12 hours.
  • the prodrug JR3.105.5 prior to and after digestion were analyzed by SDS-PAGE (FIG.8B) and the cell-based activity assay (see below).
  • Example 5 Cell-Based IL-12 Reporter assay
  • IL-12 prodrugs prior to and after activation was assayed for biological activity using a secreted alkaline phosphatase reporter cell line (HEK-Blue IL-12, InvivoGen catalog # hkb-il12).
  • the HEK-Blue IL-12 cell line was generated through the stable introduction of the genes for the human IL-12 receptor and signaling pathway into HEK 293 cells.
  • the cells also express a STAT4 inducible secreted alkaline phosphatase (SEAP) reporter gene.
  • SEAP STAT4 inducible secreted alkaline phosphatase
  • Test articles are diluted in 100 ⁇ L/well culture medium in 96 well flat bottom cell culture plates, followed by addition of HEK-Blue IL-12 cells at 50,000 cells/well in 100 ⁇ L. Cultures are incubated at 37 o C overnight. 20 ⁇ L of culture supernatant is transferred to an ELISA plate, and 180 ⁇ L Quanti-Blue (InvivoGen, Catalog #rep-qb1) is added. The ELISA plate is incubated at 37 o C for 1 hour, and OD630 is measured with a microplate reader. The data are shown in FIG.9 and FIG.10. The results show that JR3.105.5 had approximately an 18-fold activation based on the EC50 values (FIG.10).
  • IL-12 prodrug JR3.105.5 sample may have contained a small amount of the activated version of the prodrug. Consequently, the masking efficiency of the masking moiety of JR3.105.5 may have been stronger than was shown in FIGs.9 and 10.
  • This IL-12 prodrug JR3.105.5 also had the highest activation compared with the other samples.
  • the IL-12 prodrugs all had the same Fc-IL-12 chain (SEQ ID NO: 20, Table 2). However, the masking moiety of JR3.105.5 had the longest linker between its VL and VH, comprising (G 4 S) 4 , as shown SEQ ID NO: 80.
  • Example 6 In vivo Efficacy Study with a Mouse Tumor Model
  • NOD SCID gamma (NSG) mice are engrafted intradermally with 3 ⁇ 10 6 pp-65 expressing MCF-7 cells in the right flank. After the tumors are established, mice are engrafted intraperitoneally with 1.5 ⁇ 10 6 human PBMCs. Mice are then treated on twice weekly, weekly, or biweekly with the IL-12 prodrugs. Tumor volumes and body weights are measured every other day.
  • mice are sacrificed at the end of the study, and blood samples, serum samples, and tissues (tumors and major organs including livers and lungs) are analyzed for PK, safety, cytokine release, and prodrug activation.

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Abstract

La présente invention concerne de nouveaux promédicaments d'IL-12. La présente invention concerne en outre des procédés de production et d'utilisation des nouveaux promédicaments.
PCT/US2020/053155 2019-09-28 2020-09-28 Promédicaments à base de cytokine et promédicaments doubles WO2021062406A1 (fr)

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WO2024150174A1 (fr) 2023-01-11 2024-07-18 Bright Peak Therapeutics Ag Immunocytokines activées de manière conditionnelle et procédés d'utilisation
WO2024150175A1 (fr) 2023-01-11 2024-07-18 Bright Peak Therapeutics Ag Protéines activées de manière conditionnelle et procédés d'utilisation

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